RGS20 Promotes Tumor Progression through Modulating PI3K/AKT Signaling Activation in Penile Cancer.

Regulator of G protein signaling 20 (RGS20) plays an important role in regulating neuronal G protein-coupled receptor signaling; however, its expression and oncogenic function in penile cancer (PC) remains unclear. Here, we observed high RGS20 expression in PC tissues compared to normal/adjacent penile tissues, which was closely associated with tumor stage, nodal status, and pelvic metastasis in our PC cohort. The cellular functional analysis of RGS20 revealed that manipulation of the RGS20 expression markedly affected cell viability, BrdU incorporation, soft agar clonogenesis, caspase-3 activity, and cell migration/invasion in PC cell models. Moreover, RGS20 could interact with PI3K p85α subunit and regulate PI3K/AKT signaling activation in PC cell lines. Knockdown of the PI3K p85α or p110α subunit attenuated cell viability, BrdU incorporation, soft agar clonogenesis, and cell migration/invasion in PC cell lines. In contrast, the overexpression of constitutively activated PI3K p110α mutant restored cell proliferation and cell migration/invasion caused by RGS20 depletion in PC cells. Consistent with the in vitro findings, RGS20 depletion attenuated PI3K/AKT signaling activation and suppressed tumor growth in a murine xenograft model. Importantly, the high RGS20 expression was associated with PI3K/AKT signaling activation and unfavorable progression-free/overall survival, highlighting the clinical relevance of RGS20/PI3K/AKT signaling in PC. In conclusion, the aberrant RGS20 expression may serve as a diagnostic and prognostic marker for PC. RGS20 may promote PC progression through modulating PI3K/AKT signaling activation, which may assist with the development of RGS20-targeting therapeutics in the future.

Systematic Pan-Cancer Analysis of KLRB1 with Prognostic Value and Immunological Activity across Human Tumors.

INTRODUCTION: KLRB1 is a gene encoding CD161 expressed in NK cells and some T cell subsets. At present, KLRB1 is believed to affect tumorigenesis and development by regulating the cytotoxicity of NK cells in several cancers. However, there is a lack of systematic reviews of KLRB1 in a variety of malignancies. OBJECTIVES: Hence, our research is aimed at providing a relatively comprehensive understanding of the role of KLRB1 in different types of cancer, paving the way for further research on the molecular mechanism and immunotherapy potential of KLRB1. METHODS: In this study, we used relevant public databases, including TCGA (The Cancer Genome Atlas), GEO (Gene Expression Omnibus), CCLE (Cancer Cell Line Encyclopedia), GTEx (Genotype Tissue-Expression), and HPA (Human Protein Atlas), to perform a pan-cancer analysis of KLRB1 across 33 types of cancer. We explored the potential molecular mechanism of KLRB1 in clinical prognosis and tumor immunity from the aspects of gene expression, survival status, clinical phenotype, immune infiltration, immunotherapy response, and chemotherapeutic drug sensitivity. RESULTS: KLRB1 was downregulated in 13 cancers while upregulated in kidney cancer. Patients with high expression of KLRB1 have a better prognosis in most types of cancer. Moreover, the KLRB1 expression level is related to TMB and MSI and related to various immune signatures of tumor. The expression of KLRB1 can affect tumor immune cell infiltration. KLRB1 expression level can also affect the sensitivity of chemotherapy drugs. CONCLUSIONS: KLRB1 may be a prognostic and immunological biomarker across tumors. At the same time, KLRB1 expression can reflect the sensitivity of cancer patients to chemotherapy drugs. KLRB1 may become a new target for immunotherapy.

DPP4 Regulates DHCR24-Mediated Cholesterol Biosynthesis to Promote Methotrexate Resistance in Gestational Trophoblastic Neoplastic Cells.

Metabolic reprogramming could promote cellular adaptation in response to chemotherapeutic drugs in cancer cells. Herein, we aimed to characterize the metabolomic profiles regulated by Dipeptidyl Peptidase 4 (DPP4) in methotrexate (MTX)-resistant gestational trophoblastic neoplastic (GTN) cells. A total of eighty metabolites were found to be commonly altered in DPP4-depleted JAR/MTX and JEG3/MTX cells. Cholesterol biosynthesis-related metabolites were markedly impacted by DPP4 knockdown in MTX-resistant sublines. Manipulation of DPP4 expression remarkably affected the level of cellular cholesterol in GTN cells. Our analysis also identified 24-Dehydrocholesterol Reductase (DHCR24) as a potential downstream effector of DPP4. Manipulation of DHCR24 expression affected cellular cholesterol level, reactive oxygen species (ROS) accumulation, and chemosensitivity to MTX in GTN cell models. In addition, over-expression of DHCR24 could markedly restore cellular cholesterol level and rescue cell survival in DPP4-depleted MTX-resistant GTN cells. Highly correlated expression of DPP4 and DHCR24 was observed in clinical GTN specimens. Further, DPP4 inhibitor sitagliptin effectively inhibited cholesterol biosynthesis, reduced DHCR24 expression and enhanced MTX-induced cytotoxicity in vitro and in vivo. In conclusion, our findings suggested that DPP4 might regulate DHCR24-mediated cholesterol biosynthesis to promote methotrexate resistance in GTN cells. Targeting DPP4/DHCR24 signaling might help to sensitize MTX-resistant GTN to MTX treatment.

Genome Instability-Related miRNAs Predict Survival, Immune Landscape, and Immunotherapy Responses in Gastric Cancer.

BACKGROUND: Increasing evidence suggests that microRNAs (miRNAs) are involved in genome instability (GI) and drive the occurrence of tumors. However, the role of GI-related miRNAs in gastric cancer (GC) remains largely unknown. Herein, we developed a novel GI-related miRNA signature (GIMiSig) and further investigated its role in prognosis, the immune landscape, and immunotherapy responses in GC patients. METHODS: An analysis of somatic mutation data on 434 gastric cancer cases from The Cancer Genome Atlas (TCGA) database was performed, thereby generating genome stability (GS) and GI groups. By detecting differentially expressed miRNAs between the GS and GI groups that were associated with overall survival, 8 miRNAs were identified and used to construct the GIMiSig. RESULTS: The GIMiSig showed high accuracy in detecting GC patients. Using GIMiSig to stratify the patients into the high- and low-risk subgroups to predict survival outperformed the use of regular clinical features such as age, gender, or disease stage. Patients with low risk had a more favorable survival time than those with high risk. More importantly, the high-risk patients were associated with decreased UBQLN4 expression, higher accumulation of immune cells, lower Titin (TTN) mutation frequency, worse immunotherapy efficacy, and cancer-associated pathways. Conversely, the low-risk patients were characterized by UBQLN4 overexpression, lower fraction of immune cells, higher TTN mutation frequency, better response to immunotherapy, and GI-related pathways. CONCLUSION: In summary, we constructed a novel GIMiSig that could stratify GC patients into distinct risk groups that have different survival outcomes and immunotherapy efficacy. The results may provide new clues for improving GC outcomes.

miR-497-5p/SALL4 axis promotes stemness phenotype of choriocarcinoma and forms a feedback loop with DNMT-mediated epigenetic regulation.

Choriocarcinoma stem-like cells (CSLCs) might be at the origin of choriocarcinoma development associated with drug resistance or relapse. Spalt-like transcription factor 4 (SALL4), which is considered to be a stemness-related gene, can be regulated by miRNAs. In this study, SALL4 result is associated with progression-free survival of choriocarcinoma patients and CSLC's stemness characteristics. In addition, it could be downregulated by miR-497-5p by direct binding. miR-497-5p silencing by hypermethylation promoted malignant CSLC phenotype in vitro and in vivo. Furthermore, increased DNA methyltransferases (DNMTs) by SALL4 upregulation inhibited miR-497-5p expression via hypermethylation promotion. SALL4 appeared to be a key factor in promoting stemness phenotype of choriocarcinoma. Silencing miR-497-5p and SALL4 promotes choriocarcinoma progression and forms a feedback loop with DNMT-mediated epigenetic regulation, playing a crucial role in stemness maintenance in choriocarcinoma.

Development and Validation of a TNF Family-Based Signature for Predicting Prognosis, Tumor Immune Characteristics, and Immunotherapy Response in Colorectal Cancer Patients.

In this study, a comprehensive analysis of TNF family members in colorectal cancer (CRC) was conducted and a TNF family-based signature (TFS) was generated to predict prognosis and immunotherapy response. Using the expression data of 516 CRC patients from The Cancer Genome Atlas (TCGA) database, TNF family members were screened to construct a TFS by using the univariate Cox proportional hazards regression and the least absolute shrinkage and selection operator- (LASSO-) Cox proportional hazards regression method. The TFS was then validated in a meta-Gene Expression Omnibus (GEO) cohort (n = 1162) from the GEO database. Additionally, the tumor immune characteristics and predicted responses to immune checkpoint blockade in TFS-based risk subgroups were analyzed. Eight genes (TNFRSF11A, TNFRSF10C, TNFRSF10B, TNFSF11, TNFRSF25, TNFRSF19, LTBR, and NGFR) were used to construct the TFS. Compared to the high-risk patients, the low-risk patients had better overall survival, which was verified by the GEO data. In addition, a high TFS risk score was associated with high infiltration of regulatory T cells (Tregs), nonactivated macrophages (M0), natural killer cells, immune escape phenotypes, poor immunotherapy response, and tumorigenic and metastasis-related pathways. Conversely, a low TFS risk score was related to high infiltration of resting CD4 memory T cells and resting dendritic cells, few immune escape phenotypes, and high sensitivity to immunotherapy. Thus, the eight gene-based TFS is a promising index to predict the prognosis, immune characteristics, and immunotherapy response in CRC, and our results also provide new understanding of the role of the TNF family members in the prognosis and treatment of CRC.

Activation of RSK2 upregulates SOX8 to promote methotrexate resistance in gestational trophoblastic neoplasia.

Resistance to chemotherapy is frequently driven by aberrantly activated kinases in cancer. Herein, we characterized the global phosphoproteomic alterations associated with methotrexate (MTX) resistance in gestational trophoblastic neoplastic (GTN) cells. A total of 1111 phosphosites on 713 proteins were significantly changed, with highly elevated Ribosomal S6 Kinase 2 (RSK2) phosphorylation (pS227) observed in MTX-resistant GTN cells. Activation of RSK2 promoted cell proliferation and survival after MTX treatment in GTN cell models. Interestingly, RSK2 might play an important role in the regulation of reactive oxygen species (ROS) homeostasis, as manipulation of RSK2 activation affected ROS accumulation and SOX8 expression in GTN cells. In addition, overexpression of SOX8 partly rescued cell proliferation and survival in RSK2-depleted MTX-resistant GTN cells, suggesting that SOX8 might serve as a downstream effector of RSK2 to promote MTX resistance in GTN cells. Highly activated RSK2/SOX8 signaling was observed in MTX-resistant GTN specimens. Further, the RSK2 inhibitor BIX02565 effectively reduced SOX8 expression, induced ROS accumulation, and enhanced MTX-induced cytotoxicity in vitro and in vivo. Collectively, our findings suggested that RSK2 activation could promote MTX resistance via upregulating SOX8 and attenuating MTX-induced ROS in GTN cells, which may help to develop experimental therapeutics to treat MTX-resistant GTN.

Quantitative Proteomic Profiling Identifies SOX8 as Novel Regulator of Drug Resistance in Gestational Trophoblastic Neoplasia.

The development of drug resistance remains one of the major challenges to current chemotherapeutic regimens in gestational trophoblastic neoplasia (GTN). Further understanding on the mechanisms of drug resistance would help to develop more effective therapy to treat GTN. Herein, tandem mass tag-based (TMT) quantitative proteomic technique was used to establish drug resistance-related proteomic profiles in chemoresistant GTN cell models (JEG3/MTX, JEG3/VP16, JEG3/5-Fu). In total, we identified 5,704 protein groups, among which 4,997 proteins were quantified in JEG3 and its chemoresistant sublines. Bioinformatics analysis revealed that multiple biological processes/molecular pathways/signaling networks were involved in the regulation of drug resistance in chemoresistant JEG3 sublines. SOX8 was upregulated in all the three chemoresistant sublines, and its function was further investigated. Knockdown of SOX8 significantly reduced cell viability, impaired soft agar clonogenesis, and increased caspase-3 activities after drug treatment in JEG3 chemoresistant sublines. In addition, over-expression of SOX8 promoted cell survival, enhanced soft agar clonogenesis, and attenuated caspase-3 activities after drug treatment in GTN cells. Importantly, SOX8 might be a potential regulator of reactive oxygen species (ROS) homeostasis, as SOX8 regulated the expression of antioxidant enzymes (GPX1, HMOX1) and reduced drug-induced ROS accumulation in GTN cell models. Collectively, SOX8 might promote drug resistance through attenuating the accumulation of ROS induced by chemotherapeutic drugs in GTN cells. Targeting SOX8 might be useful to sensitize GTN cells to chemotherapy.

Quantitative proteomic analysis identifies novel regulators of methotrexate resistance in choriocarcinoma.

OBJECTIVE: Although methotrexate (MTX) is commonly used for the treatment of choriocarcinoma, chemoresistance to MTX may occur in a considerable fraction of patients. Further understanding on the mechanisms of MTX resistance would help to develop more effective therapy for choriocarcinoma. METHODS: Quantitative proteomic approach involving TMT labeling and LC-MS/MS was used to identify MTX resistance-related proteomic profiles in choriocarcinoma cell models. Pathway and process enrichment analysis were conducted to identify MTX resistance-related biological processes/molecular pathways. CCK-8 viability assay, clonogenic survival assay, and BrdU incorporation analysis were used to examine the chemosensitivity to MTX in choriocarcinoma cells. RESULTS: In total, 5704 protein groups were identified, among which 4997 proteins were quantified. Bioinformatic analysis revealed that multiple biological processes/molecular pathways might be associated with MTX resistance in JEG3/JEG3/MTX cell systems. DPP4 and METTL7A were selected for further investigation. Increased expression of DPP4 or METTL7A was observed in MTX-resistant cancer cell lines and choriocarcinoma tissues. Knockdown of DPP4 or METTL7A significantly decreased cell viability, impaired clonogenesis, and increased apoptosis after MTX treatment in JEG3/MTX and JAR/MTX cells; while over-expression of DPP4 or METTL7A promoted cell viability and reduced apoptosis following exposure to MTX in JEG3, JAR and BEWO cells. Further, DPP4 and METTL7A differentially activated prosurvival signaling pathways including PI3K/AKT, ERK1/2 and STAT3, and attenuated the accumulation of reactive oxygen species (ROS) in choriocarcinoma cell lines. CONCLUSIONS: DPP4 and METTL7A might promote MTX resistance through activating pro-survival signaling pathways and attenuating the accumulation of ROS in choriocarcinoma cells. Targeting DPP4 and METTL7A might be useful to sensitize choriocarcinoma cells to MTX-based chemotherapy.

SLAMF1 Promotes Methotrexate Resistance via Activating Autophagy in Choriocarcinoma Cells.

OBJECTIVE: The acquisition of chemoresistance to methotrexate (MTX) still remains one of the major challenges for choriocarcinoma treatment. Herein, we aimed to evaluate the potential role of Signaling Lymphocytic Activation Molecule Family Member 1 (SLAMF1) as a possible regulator of chemoresistance to MTX in choriocarcinoma. MATERIAL AND METHODS: MTX-resistant JEG3 and JAR sublines (JEG3/MTX, JAR/MTX) were used to study SLAMF1 function. CCK8 assay and soft agar assay were conducted to measure the cell viability and clonogenesis of choriocarcinoma cells, respectively; MDC incorporation assay was conducted for the quantification of intracellular autophagy; BrdU labeling was used to assess the proliferative potential of choriocarcinoma cells; SLAMF1 protein expression was analyzed by Western blotting. RESULTS: Upregulation of SLAMF1 expression was observed in MTX-resistant JEG3/MTX and JAR/MTX sublines compared to their parental JEG3 and JAR cell lines, respectively. Knockdown of SLAMF1 markedly attenuated cell viability and soft agar clonogenesis after incubation with MTX in JEG3/MTX and JAR/MTX cells. In contrast, constitutive expression of SLAMF1 rescued cell survival soft agar clonogenesis in JEG3 and JAR cells treated with MTX. Moreover, autophagy is apparently activated in MTX-resistant JEG3/MTX and JAR/MTX sublines compared to their parental cell lines. Autophagy inhibitor 3-methyladenine and bafilomycin A1 enhanced MTX-induced cytotoxicity in MTX-resistant JEG3 and JAR sublines. Further, SLAMF1 might activate autophagy-related mechanism to promote resistance to MTX in choriocarcinoma cells. Depletion of SLAMF1 suppressed autophagy and induced apoptosis in MTX-treated JEG3/MTX and JAR/MTX cells. CONCLUSION: SLAMF1 might promote MTX resistance via activating protective autophagy in choriocarcinoma cell lines. Targeting SLAMF1 might be a useful therapeutic strategy to sensitize choriocarcinoma cells to MTX-based regimens.

The long non-coding RNA MALAT1 interacted with miR-218 modulates choriocarcinoma growth by targeting Fbxw8.

Among the first found cancer-related long non-coding RNAs (lncRNAs), MALAT1 is one that involves in the development and progression of some tumors. MALAT1 can be aberrantly expressed in hepatocellular carcinoma, cervical, breast, ovarian cancers, as well as colorectal cancer. The paper aims to make certain the function of MALAT1 in human choriocarcinoma cell lines by investigating the detailed effects and molecular mechanisms. Being specifically upregulated in choriocarcinoma cell lines, the under-researched lncRNA-MALAT1 promoted choriocarcinoma cell growth by targeting miR-218. After MALAT1 knockdown, proliferation of human choriocarcinoma cell in vitro was dramatically hindered, and the tumor size in vivo was reduced. What is more, miR-218-mediated Fbxw8 regulation was required for MALAT1-induced choriocarcinoma cell proliferation. Taken together, MALAT1 might promote choriocarcinoma tumor growth through miR-218-mediated Fbxw8 regulation. According to our data, MALAT1 might be an oncogenic lncRNA that promoted choriocarcinoma proliferation and could be therapeutically targeted in human choriocarcinoma.

MicroRNA-218 inhibits the proliferation of human choriocarcinoma JEG-3 cell line by targeting Fbxw8.

MicroRNAs (miRNAs) are endogenous 19-25 nucleotide noncoding single-stranded RNAs that regulate gene expression by blocking the translation or decreasing the stability of mRNAs. In this study, we showed that miR-218 expression levels were decreased while Fbxw8 expression levels were increased in human choriocarcinoma cell lines, and identified Fbxw8 as a novel direct target of miR-218. Overexpression of miR-218 inhibited cell growth arrest at G2/M phase, suppressed the protein levels of cyclin A and up-regulated the expression levels of p27 through decreasing the levels of Fbxw8. On the other hand, forced expression of Fbxw8 partly rescued the effect of miR-218 in the cells, attenuated cell proliferation decrease the percentage of cells at G2/M phase, induced cyclin A protein expression and suppressed the protein level of p27 through up-regulating the levels of Fbxw8. Taken together, these findings will shed light the role to mechanism of miR-218 in regulating JEG-3 cells proliferation via miR-218/Fbxw8 axis, and miR-218 may serve as a novel potential therapeutic target in human choriocarcinoma in the future.

Ultrasound elastography in the differential diagnosis of benign and malignant cervical lesions.

OBJECTIVES: This study aimed to evaluate the clinical value of ultrasound elastography in the differential diagnosis of benign and malignant cervical lesions and to compare the accuracy of the elasticity score and strain ratio in differentiating cervical lesions. METHODS: B-mode sonography and ultrasound elastography were performed on 84 cervical lesions (40 benign and 44 malignant) in 84 patients. All of the images were obtained transvaginally. The elasticity score was determined by a 5-point scoring method. Calculation of the strain ratio was based on a comparison of the average strain measured in the lesion with the adjacent tissue of the same depth, size, and shape. The findings were compared with histopathologic results. With the use of receiver operating characteristic curves, the diagnostic value of the elasticity score and strain ratio methods was determined. RESULTS: The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of the elasticity score in the differential diagnosis of cervical lesions were 81.8%, 85.0%, 83.3%, 85.7%, and 81.0%, respectively, whereas those of the strain ratio were 90.9%, 90.0%, 90.5%, 90.9%, and 90.0%. A strain ratio cutoff value of 4.525 was used as a standard to distinguish benign from malignant lesions. The strain ratio values of malignant lesions were much higher than those of benign lesions (range, 4.85-8.91 versus 0.62-4.50). The differences were statistically significant (P < .01). CONCLUSIONS: Ultrasound elastography is a promising technique that is easy and rapid to perform and can help identify cervical lesions that are likely to be malignant. It is obvious that the strain ratio yielded better results than the elasticity score. Both methods are semiquantitative, but quantification of the strain ratio is finer than that of the elasticity score.